Polymerase chain reaction, i.e. PCR technique, is a method for rapidly amplifying a specific gene or DNA sequence in vitro, which was invented by the scientist K. B. Mullis from Department of Human Genetics at Cetus Corporation on 1983. PCR utilizes a thermotolerant DNA polymerase, mixes primers and a target DNA to massively amplify the target DNA in short time through cycles consisting of high temperature denaturation, low temperature annealing and appropriate temperature extension. PCR technique has advantages such as high specificity, sensitivity and yield, rapidity, simplicity, good repeatability, facilitation in automation. PCR technique can amplify a target gene or certain DNA fragment to be studied ten thousands and even million times in a tube in several hours, enabling direct observation and determination of the result with the naked eye. PCR technique can amplify sufficient amount of DNA from a hair, a drop of blood, even a cell for analysis, research, detection and identification. PCR technique is a revolutionary innovation and a landmark in the field of biological medicine, but it has no way to get rid of the restriction of thermal cycling. After a few decades of development, some isothermal nucleic acid amplification techniques are gradually invented.
The nucleic acid isothermal amplification technique is a technique that a target gene is massively replicated at constant temperature. At present, the methods for nucleic acid isothermal amplification mainly comprise the following methods. TAS, i.e. Transcript-based Amplification System, was developed in 1989 by SISKA Diagnostic Institute and Salk Institute from the United States; it is mainly applied for RNA amplification with a reaction principle that a target RNA is massively amplified under an action of reverse transcriptase and T7 RNA polymerase through multistep temperature change. During the amplification process, it mainly depends on the polymerase activity of the reverse transcriptase and the activity of RNase H, such amplification manner gets rid of the limitation of high temperature cycling, and only needs to set multistep temperature. The method is of greatly progressive significance, has high specificity and sensitivity. However, the reverse transcriptase and T7 RNA polymerase are required to be added continually during the reaction, which brings a great deal of inconvenience to use. Moreover, it can only be used for RNA amplification currently.
NASBA, i.e. Nucleic Acid Sequence-Based Amplification, was first introduced by Canadian Can-gene Corporation in 1991 in an article. NASBA is an isothermal amplification technique which can massively replicate nucleic acid sequence in vitro. NASBA is a stable and sustained enzymatic reaction medicated by two primers. NASBA reaction relies on AMV reverse transcriptase, RNase H, T7 RNA polymerase, the complete reaction is performed at the constant temperature (41° C.), and a desirable result may be obtained in 1.5-2 h. The drawbacks of NASBA are that there still requires a complex sequential procedure for products detection; the enzymes are not thermotolerant, and can be added only after the RNA chain is melted; nonspecific interaction of the primers due to low temperature causes nonspecific amplification; three enzymes are required to be added into the reaction and to be activated in the same reaction system at the same temperature.
3SR, i.e. Self-Sustained Sequence Replication, is also developed by SISKA Diagnostic Institute and Salk Institute of the United States based on TAS. It has a principle substantially similar to that of TAS, except that RNase H is needed, and, the reaction also requires AMV reverse transcriptase and T7 RNA polymerase. Compared to NASBA, 3SR is more complex, and its sensitivity is not high. NASBA is gradually replacing 3SR.
SDA, i.e. Strand Displacement Amplification, was established by American scholar Wallker, et al in 1992. It utilizes the capability of restriction enzyme of shearing DNA recognition site and that of DNA polymerase of extending from the nick to 3′ end and substituting the downstream sequence to massively amplify a target sequence under isothermal condition. The drawbacks of SDA are that there still a need for denaturation process, the target sequence to be amplified cannot exceed 200 bp, and the follow-on detection is complex.
RCA, i.e. Rolling Circle Amplification, was invented in 1998. The inspiration of this method mainly derives from the replication process of a circular DNA of a microorganism in the nature, which is invented by simulating amplification of such circular DNA in vitro. The method can massively amplify a target sequence mainly with DNA polymerase by ingenious primer design. However, its amplification products are very complicated, and have various sizes of the fragments. Since RCA can only amplify a circular DNA template, its application range is greatly restricted.
HDA, i.e. Helicase-Dependent Amplification, is an in vitro isothermal nucleic acid amplification technique, first introduced by researchers Vincent, et al. from New England Biolabs Incorporation of the United States in 2004. Its reaction principle lies in that the double stranded DNA was opened under the action of a helicase, and a single strand DNA binding protein binds the template single strand, which makes the DNA keep a single strand state, then primers bind to the single strand for extending forward under the action of DNA polymerase. However, HAD amplification also requires three enzymes, which greatly limits its application scope.
SPIA, i.e. Single Primer Isothermal Amplification, was invented in 2005. With the technique, a single stranded cDNA is massively amplified by a hybrid with a DNA fragment at 3′ end and a RNA fragment at a 5′ end under actions of RNase H and DNA polymerase having strong strand displacement activity. SPIA amplification is performed at a temperature of about 60° C., and completed in half of an hour. SPIA amplification technique has the advantages of high amplification efficiency, strong conservation, simple principle, etc, and is adapted for nucleic acid detection, nucleic acid sequencing, SNP detection, single stranded template preparation and gene chip probe preparation, etc.
A novel in vitro nucleic acid amplification technique, i.e. Loop-mediated Isothermal Amplification (LAMP), was established by Notomi, et al. in 2000. Such novel amplification method requires at least 4 primers, at most 6 primers to specificity identify 6, 7 or 8 regions of a target fragment, and amplifies the target sequence under the action of chain displacement of a DNA polymerase in short time. Such technique has properties of simplicity, specificity, efficiency and rapidity. When the target DNA is massively synthesized, byproduct, i.e. white magnesium pyrophosphate precipitate, is produced, which makes LAMP reaction be directly judged as a negative or positive result by a change of turbidity. PCR requires two primers to specifically bind two sections of a target sequence, while LAMP specifically binds 6 to 8 sections, having stronger specificity than PCR. LAMP is 10-100 fold more sensitive than common PCR, which is comparable to fluorescent quantitative PCR. Moreover, LAMP amplification is performed in isothermal condition, a thermostable device (such as an isothermal water bath kettle, an isothermal metal bath, etc) can meet the reaction requirement, thereby greatly reducing detection cost. Since LAMP technique was reported, it has been widely applied to detection of microorganisms such as bacterium, fungus, virus, etc, diagnosis of genetic disease, and early determination of gender in a short period of more than ten years.
The results of comparison of various isothermal amplification techniques are shown in Table 1 for detail.
TABLE 1The results of comparison of variousisothermal amplification techniquesNumber ofInventionenzymesApplicationNametimerequiredrangePubMed※Transcript-based19892RNA44AmplificationSystem (TAS)Self-Sustained19903DNA and RNA245SequenceReplication (3SR)Nucleic Acid19913DNA and RNA2782Sequence-BasedAmplification(NASBA)Strand19923DNA and RNA267DisplacementAmplification(SDA)Rolling Circle19981circular DNA541Amplificationand RNA(RCA)Loop-mediated20001DNA and RNA934IsothermalAmplification(LAMP)Helicase-Dependent20043DNA99Amplification(HDA)Single Primer20053DNA and RNA137IsothermalAmplification(SPIA)※Note:PubMed represents the number of articles searched on PubMed in term of English full name of various isothermal amplificationtechniques, by the end of Mar. 25, 2014.LAMP is predominant in these isothermal nucleic acid amplification methods, but the LAMP products are too complex, and recovery and sequencing of the LAMP products are very difficult, they cannot be directly sequenced like common PCR products. Since LAMP products are the amplification mixture that is extremely complex and irregular, LAMP products cannot be used for cloning. These defects make LAMP be limited in gene rapid detection.